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1.
Journal of Experimental Hematology ; (6): 66-69, 2012.
Article in Chinese | WPRIM | ID: wpr-331020

ABSTRACT

This study was aimed to evaluate the effect of triptolide (TPL) on the reversal of multidrug resistance in K562/A02 cell line. The sensitivity of K562 and K562/A02 to adriamycin (ADM) and reversal of drug resistance were determined with MTT method. The concentration of intracellular ADM and P-glycoprotein expression were detected by flow cytometry. Luciferase reporter gene assay was used to detect the transcriptional activity of MDR1 promoter. The results showed that TPL significantly decreased the resistance degree of K562/A02 cells, inhibited P-glycoprotein expression (mean fluorescent intensity decreased from 123 ± 13 to 39 ± 13) and increased the intracellular concentration of ADM (mean fluorescent intensity increased from 18 ± 5 to 34 ± 6) in K562/A02 cells. Luciferase reporter gene assay demonstrated that TPL inhibited the transcriptional activity of MDR1 promoter by 75%. It is concluded that TPL may effectively reverse the multidrug resistance in K562/A02 cells via modulating P-glycoprotein expression and increasing intracellular ADM accumulation.


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Diterpenes , Pharmacology , Doxorubicin , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Epoxy Compounds , Pharmacology , K562 Cells , Phenanthrenes , Pharmacology , Promoter Regions, Genetic
2.
Journal of Experimental Hematology ; (6): 34-39, 2011.
Article in Chinese | WPRIM | ID: wpr-244990

ABSTRACT

This study was aimed to explore the effect of vascular endothelial growth factor (VEGF) on sensitivity of leukemia cell line K562/A02 to doxorubicin by using RNA interference, and to investigate its mechanism. The 3 shRNA targeting human vegf gene were synthesized, then transfected into K562/A02 cells by lipofectamine 2000 reagent. RT-PCR was used to detect the expression of vegf and mrp1 at the mRNA level;Western blot was used to analyze the expression of VEGF, MRP1, AKT, P-AKT at the protein level; MTT was used to determine the IC(50) value of transfected cells to doxorubicin (DOX); flow cytometry was used to detect cell apoptosis and intracellular Rho123 retention. The results showed that after vegf shRNA were transfected into K562/A02 cells, the expression of vegf at the mRNA level decreased, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was statistically significant (p < 0.05), the greatest decrease was observed in the cells transfected with vegf shRNA3; and the protein level of VEGF was also down-regulated. The IC(50) value of positively transfected group was lower than that of control groups, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was significant (p < 0.05). The retention of intracellular Rho123 was enhanced in three positively transfected groups (p < 0.05). Cell apoptosis increased in positively transfected groups, and there was statistically difference between vegf shRNA2 group or vegf shRNA3 group and HK group (p < 0.05). The expression of mrp1 at the mRNA level were decreased, and there were statistical difference between vegf shRNA3 group and HK group (p < 0.05), and the protein level of mrp1 was also down-regulated; the expression of P-AKT at protein level decreased in positively transfected groups, and the greatest decrease was seen in vegf shRNA3 group. It is concluded that the transfection with exogenous vegf shRNA can inhibit the expression of vegf at both mRNA and protein levels, and enhance the sensitivity of K562/A02 cell to doxorubicin, the mechanism of which may be the inhibition of apoptosis and down-regulation of MRP1 by inactivating PI3K/AKT signaling pathway.


Subject(s)
Humans , Apoptosis , Doxorubicin , Pharmacology , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , K562 Cells , Multidrug Resistance-Associated Proteins , Genetics , RNA Interference , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Transfection , Vascular Endothelial Growth Factor A , Genetics
3.
Chinese Journal of Hematology ; (12): 383-387, 2011.
Article in Chinese | WPRIM | ID: wpr-251945

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of YB-1 on the transcription of induced mdr1 gene expression in K562 cells.</p><p><b>METHODS</b>K562 cells were treated with doxorubicin (DOX) at different concentrations and times. Expression of mdr1 and YB-1 genes was examined by RT-PCR and P-glycoprotein (P-gp) by flow cytometry. Cyto/nuclear protein was extracted for YB-1 detection by Western blotting. The expression of YB-1 gene in K562 cells was inhibited by YB-1 gene specific RNA interference (RNAi), then the expression of mdr1 and P-gp in YB-1 gene silenced cells treated with DOX was detected.</p><p><b>RESULTS</b>The mdr1 gene as well as its corresponding protein P-gp was highly expressed in DOX exposed K562 cells. DOX up-regulated the expression of YB-1 gene, and promoted YB-1 protein nuclear translocation. On YB-1 gene silenced, the expressions of mdr1 gene and P-gp were obviously down-regulated in DOX treated K562 cells.</p><p><b>CONCLUSION</b>Doxorubicin can induce the expression of mdr1 gene in K562 cells, which may result from the transcription of mdr1 gene by activated YB-1.</p>


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Doxorubicin , Pharmacology , Drug Resistance, Neoplasm , Genetics , Gene Expression , Gene Silencing , K562 Cells , Protein Transport , RNA Interference , RNA, Small Interfering , Y-Box-Binding Protein 1 , Genetics
4.
Journal of Experimental Hematology ; (6): 337-341, 2011.
Article in Chinese | WPRIM | ID: wpr-244926

ABSTRACT

Iron is an essential element for cell growing including tumor cells. This study was purposed to explore the effect of desferrioxamine (DFO) on cell line K562/A02 and its mechanism. K562/A02 cells were cultured with different concentrations of DFO. The inhibitory effects of adriamycin (ADM) used alone or combined with DFO on the proliferation of K562/A02 was evaluated by MTT assay. The apoptosis rate of K562/A02 cells after treatment with 0, 12.5, 25 and 50 µmol/L DFO alone or in combination with 1 mg/L ADM were analyzed by flow cytometry. ADM accumulation in K562/A02 cells after treatment with different concentrations of 0, 12.5, 25 and 50 µmol/L DFO were also analyzed by flow cytometry. The levels of BAX/BCL-2 and MDR1 mRNA were determined by RT-PCR, and then the protein level of P-glycoprotein (P-gp) was detected by Western blot. The results showed that the IC(50) of ADM for K562 and K562/A02 cells were (1.46 ± 0.07) mg/L and (40.98 ± 3.05) mg/L respectively. The resistance of K562/A02 cells to ADM was 28.06 times as that of K562 cells. After treatment of K562/A02 cell with DFO of 12.5, 25 and 50 µmol/L for 48 hours, the resistance of K562/A02 cells to ADM were increased by 24.95, 16.11 and 9.99 times respectively. When K562/A02 cells were incubated with different concentrations of DFO of 12.5, 25, 50 µmol/L for 48 hours, the apoptosis rat were (3.50 ± 0.30)%, (7.27 ± 0.32)% and (12.53 ± 1.21)% respectively. After co-culture with DFO and ADM for 48 hours, apoptosis rate were (6.13 ± 0.29)%, (9.57 ± 0.40)% and (18.97 ± 1.10)% respectively. The above apoptosis rates was much higher than that of control group (p < 0.05) and they were dose-dependent. In comparison between DFO + ADM group and DFO group, there was no significant difference (p > 0.05). Expression rate of BAX/BCL-2 increased. The levels of MDR1 mRNA reduced. Furthermore, expression of P-gp also decreased in K562/A02 cells. It is concluded that iron increase can promote K562/A02 cells growth and inhibit their apoptosis. Otherwise, iron-deprivation can induce K562/A02 cells apoptosis. DFO disturbs the iron metabolism and inhibits DNA synthesis of K562/A02 cells. This action of DFO may enhance the susceptibility of K562/A02 cells to apoptosis induced by chemotherapeutic drugs. The iron-deprivation may play a role in the treatment of leukemia with combination of DFO with other anticancer agents.


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Apoptosis , Deferoxamine , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Iron , Metabolism , K562 Cells , Proto-Oncogene Proteins c-bcl-2 , Metabolism , bcl-2-Associated X Protein , Metabolism
5.
Journal of Experimental Hematology ; (6): 342-347, 2011.
Article in Chinese | WPRIM | ID: wpr-244925

ABSTRACT

The aim of this study was to investigate whether the growth, apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells, and its possible molecular mechanisms. The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation, and the positive clones were screened by G418. RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells, respectively. The proliferative ability of the cells was determined by MTT assay and cell cycle analysis. Apoptosis of K562/A02 cells was assayed by AnnexinV-FITC/PI double labeled flow cytometry. The drug sensitivity to anticancer agent was determined by MTT assay. The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively. The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells. The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were (65.1 ± 2.1)%, (27.4 ± 1.3)% and (67.4 ± 1.6)% respectively. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells, and displayed higher at G(1), lower at G(2) and S phase in cell cycle distribution in comparison with the control groups. AnnexinV/PI detection indicated higher AnnexinV(+) ratio in 3 groups of positively transfected cells after being treated with As(2)O(3) of 0.5 µmol/L for 24 hours. The IC(50) values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group. The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells. It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis. The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Apoptosis , Cell Proliferation , Genetic Vectors , K562 Cells , RNA, Small Interfering , Genetics , Transfection , Y-Box-Binding Protein 1 , Genetics , Metabolism
6.
Journal of Experimental Hematology ; (6): 363-366, 2011.
Article in Chinese | WPRIM | ID: wpr-244921

ABSTRACT

This study was purposed to explore the mechanisms of preventive effect of tetrandrine (TTD) on doxorubicin (ADM)-induced multidrug resistance (MDR) in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis. The experiment was divided into 3 groups: group I-blank control; group II-ADM-induced drug-resistance; group III-ADM-induced drug-resistance after pretreatment with TTD. Reverse transcription-PCR (RT-PCR) was used to detect the mRNA expression levels of c-Jun, YB-1 and Survivin genes. Western blot was used to determine the nuclear protein expression levels of c-Jun and YB-1. Flow cytometry was used to assay the apoptosis of cells. The results showed that as compared with group I, the expression levels of c-Jun mRNA and nuclear protein decreased (p < 0.05), as well as the expression levels of YB-1 mRNA and nuclear protein increased in group II (p < 0.05). However, the expression of Survivin mRNA had no change (p > 0.05); the apoptosis rate of cells was 8.31%. As compared with group II, the expression levels of c-Jun mRNA and nuclear protein increased (p < 0.05), expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group III (p < 0.05). The apoptosis of cells was 97.2%. It is concluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun, thus inhibit the expression of MDR1 gene. TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Apoptosis , Genetics , Benzylisoquinolines , Pharmacology , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , Inhibitor of Apoptosis Proteins , Metabolism , K562 Cells , Proto-Oncogene Proteins c-jun , Metabolism , Y-Box-Binding Protein 1 , Metabolism
7.
Chinese Medical Journal ; (24): 2222-2227, 2011.
Article in English | WPRIM | ID: wpr-338483

ABSTRACT

<p><b>BACKGROUND</b>Recent studies have discovered that nuclear translocation of nerve growth factor (NGF) and its receptor fragments function differently from the traditional model. This study aimed to uncover the nuclear expression of NGF in astrocytoma and its biological significance.</p><p><b>METHODS</b>Ninety-four paraffin-embedded astrocytoma specimens were subjected to immunohistochemical (IHC) and hemotoxylin & eosin (HE) staining. Preoperative cerebrospinal fluid (CSF) specimens and intraoperative snap-frozen astrocytoma tissues were assayed for NGF expression by ELISA and Western blotting. The outcome of patients who contributed samples was tracked. Each ten tissue samples from patients with traumatic brain injury who had received decompression surgery and CSF samples from patients undergoing spinal anesthesia but with no history of nervous system disease were taken as control.</p><p><b>RESULTS</b>NGF-positive immunoreactive products were distributed in both the cytoplasm and nucleus of astrocytoma, but were only located in the cytoplasm of traumatic brain injury (TBI) tissue. NGF nuclear-positive rate (NPR) of grades III - IV astrocytomas (70.0%) was higher than that of grades I - II astrocytoma (28.6%, P < 0.05). NGF-NP expression positively correlated with the NGF concentration in cerebrospinal fluid (CSF) (r = 0.755, P < 0.01). Kaplan-Meier survival analysis indicated that the median survival time was 25 months for NGF-NP astrocytoma grade I - II patients and 42 months in NGF nuclear negative (NGF-NN) astrocytoma grade I - II patients (P < 0.05). In astrocytoma III - IV patients, the median survival was 7 months for NGF-NP patients and 24 months for NGF-NN patients (P < 0.01). Two types of NGF with molecular weights of 13 and 36 kDa were present in astrocytoma, but only the 36 kDa NGF was found in the CSF. NGF expression elevated as the malignancy increased.</p><p><b>CONCLUSIONS</b>NGF-NP expression and NGF level in CSF were significant prognostic factors in astrocytoma patients. Because of the easy access of CSF, it may be developed as an index for early diagnosis and surveillance of astrocytoma.</p>


Subject(s)
Female , Humans , Male , Middle Aged , Astrocytoma , Metabolism , Biomarkers , Metabolism , Blotting, Western , Cerebrospinal Fluid , Metabolism , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Nerve Growth Factor , Metabolism , Prognosis
8.
Journal of Experimental Hematology ; (6): 24-27, 2011.
Article in Chinese | WPRIM | ID: wpr-332293

ABSTRACT

The aim of this study was to investigate the potential benefit of combination therapy with 5-bromotetrandrine (5-BrTet) and daunorubicin (DNR) on chronic leukemia. The apoptosis of K562/A02 cells treated by DNA, BrTet and BrTet combined with DNR for 48 hours was detected by flow cytometry; the expressions levels of survivin mRNA and protein K562/A02 cells treated by DNR, BrTet and BrTet combined with DNR and in untreated K562 cells for 48 hours were measured by RT-PCR and Western blot respectively. The results showed that the combination of BrTet with DNR increased apoptotic rate of K562/A02, down-regulated the expression levels of survivin mRNA and protein in K562/A02 cells as compared with blank control and cells treated by BrTet or DNR alone, the survivin expression in K562/A02 cells was higher than that in K562 cells. It is concluded that the combination of BrTet with DNR can effectively reverse the multidrug resistance of K562/A02 cells, promote the apoptosis of K562/A02 cells, the mechanism of which may be related with down-regulation of survivin expression. Survivin may be a target for the treatment of MDR in hematopoietic malignancies.


Subject(s)
Humans , Apoptosis , Genetics , Benzylisoquinolines , Pharmacology , Daunorubicin , Pharmacology , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , Gene Expression Regulation, Leukemic , Inhibitor of Apoptosis Proteins , Genetics , K562 Cells
9.
Chinese Journal of Oncology ; (12): 22-28, 2010.
Article in Chinese | WPRIM | ID: wpr-295191

ABSTRACT

<p><b>OBJECTIVE</b>To evaluate the impact of a new CD44 variant on invasion of human breast cancer cell line MCF-7, and its possible mechanisms.</p><p><b>METHODS</b>The full length cDNA encoding CD44v17 was obtained from the total RNA isolated from the MCF-7/ADR cells by reverse transcript-polymerase chain reaction (RT-PCR) and subcloned into pMD19-T vector. The CD44v17 gene sequence and reading frame were confirmed by two restriction enzymes and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44v17 was transfected into MCF-7 cells by Lipofectamine. The changes of MMP-2 and MMP-9 expression at gene and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells through the artificial matrix membrane in every group was counted to compare the change of the invasive ability regulated by CD44 variant. The ERK and p-ERK were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by CD44 variant.</p><p><b>RESULTS</b>The new gene sequence was successfully cloned into recombinant vector pcDNA3.1 and identified by the two restriction enzymes. It was confirmed that the reconstructed plasmid contained the sequence of CD44 gene variant which was composed of 1 to 4 exons, 16 to 17 exons, and 1 to 205 bases of 18 exons. The new gene sequence was sent to NCBI for publication and obtained the registered number FJ216964. The up-regulated levels of the CD44 gene mRNA and protein were respectively detected by RT-PCR and flow cytometry in MCF-7 cells transfected with pcDNA3.1-CD44v17. The invasiveness of the cells and the activity of MMP-2 and MMP-9 were clearly activated by hyaluronic acid (HA) treatment and blocked by CD44 neutralizing antibody. Pretreated MCF-7/CD44v17 cells with the neutralizing antibody against CD44 and the inhibitor of MAPKs signaling pathway strongly block the expression of p-ERK.</p><p><b>CONCLUSION</b>A new CD44 gene variant has been found in adriamycin-resistant human breast cancer MCF-7/ADR cells. The expression vector pcDNA3.1-CD44v17 has been cloned and constructed successfully. HA can be integrated with CD44 variant and then regulates the expression of MMP-2 and MMP-9, which increases the invasion ability of MCF-7 cells through the Ras/MAPK signaling pathway.</p>


Subject(s)
Humans , Breast Neoplasms , Genetics , Metabolism , Pathology , Cell Line, Tumor , Doxorubicin , Pharmacology , Drug Resistance, Neoplasm , Extracellular Signal-Regulated MAP Kinases , Metabolism , Genetic Vectors , Hyaluronan Receptors , Genetics , Metabolism , Hyaluronic Acid , Pharmacology , Matrix Metalloproteinase 2 , Genetics , Metabolism , Matrix Metalloproteinase 9 , Genetics , Metabolism , Neoplasm Invasiveness , Phosphorylation , Plasmids , Protein Isoforms , RNA, Messenger , Metabolism , Recombinant Proteins , Genetics , Metabolism , Signal Transduction , Transfection
10.
Chinese Journal of Cancer ; (12): 591-595, 2010.
Article in English | WPRIM | ID: wpr-296384

ABSTRACT

<p><b>BACKGROUND AND OBJECTIVE</b>Research has shown that 5-bromotetrandrine (BrTet) can effectively reverse multidrug resistance (MDR). Imatinib plays an important role in cell proliferation. This study explored the efficacy of the combination of imatinib and BrTet on reversing MDR of tumor cells and its mechanism.</p><p><b>METHODS</b>Cytoxicity was assessed by MTT assay. Apoptosis of K562/A02 cells was analyzed by flow cytometry. The expressions of mdr1 mRNA and P-glycoprotein (P-gp) were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis.</p><p><b>RESULTS</b>After 48 h of treatment with 0.0625 micromol/L imatinib, 0.5 micromol/L BrTet, or both, the 50% inhibition concentration (IC50) of daunorubicin (DNR) for the K562/A02 cells were 5.69 mg/L, 5.41 mg/L, and 2.19 mg/L, respectively. The gray-scale values of mdr1 mRNA expression in the K562/A02 cells were 0.65+/-0.02, 0.64+/-0.01, and 0.25+/-0.03, respectively. The expression levels of P-gp were 0.74+/-0.02, 0.52+/-0.02, and 0.29+/-0.02, respectively. All decreased significantly in the K562/A02 cells treated with both imatinib and BrTet compared to cells treated with imatinib and BrTet alone (P<0.05). The apoptosis rates of the K562/A02 cells increased without a significant difference after treatment with DNR, imatinib, or BrTet (P>0.05), while increased significantly after treatment with DNR combined with imatinib, BrTet, or both (P<0.05).</p><p><b>CONCLUSIONS</b>The MDR of K562/A02 cells may be partially reversed by imatinib or BrTet, and the mechanism may be related to the downregulation of mdr1 mRNA and P-gp expression and the upregulation of the rate of apoptosis in K562/A02 cells. Imatinib combined with BrTet showed a synergistic effect on K562/A02 cells.</p>


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Metabolism , Antibiotics, Antineoplastic , Pharmacokinetics , Pharmacology , Antineoplastic Agents , Pharmacology , Apoptosis , Benzamides , Benzylisoquinolines , Pharmacology , Cell Proliferation , Daunorubicin , Pharmacokinetics , Pharmacology , Down-Regulation , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Synergism , Gene Expression Regulation, Leukemic , Imatinib Mesylate , K562 Cells , Piperazines , Pharmacology , Pyrimidines , Pharmacology , RNA, Messenger , Metabolism
11.
Journal of Experimental Hematology ; (6): 67-73, 2010.
Article in English | WPRIM | ID: wpr-328570

ABSTRACT

This study was aimed to explore the potential therapy of Gambogic acid (GA) combined with magnetic nanoparticle of Fe3O4 (Fe3O4-MNP) on leukemia. The proliferation of U937 cells and the cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscopy and flow cytometry respectively. The expressions of gene and protein were detected by quantitative real-time polymerase chain reaction and Western blot respectively. The results showed that GA enhanced the cytotoxicity for U937 cells in dose- and time-dependent manners. The Fe3O4-MNP itself had not cytotoxicity, but could enhance the inhibitory effect of GA on proliferation of U937 cells. The apoptotic rate of U937 cells induced by combination of GA with Fe3O4-MNP was higher than that by GA alone. The typical apoptotic features of cells treated with GA and Fe3O4-MNP were observed. The expression levels of caspase-3 and bax after co-treatment of GA and Fe3O4-MNP were higher than that exposed to GA or Fe3O4-MNP alone, but the expressions of bcl-2, NF-kappaB and survivin were down-regulated. It is concluded that Fe3O4-MNP can promote GA-induced apoptosis in U937 cells, and the combination of GA with Fe3O4-MNP may be a safer and less toxic new therapy for leukemia.


Subject(s)
Humans , Apoptosis , Iron Compounds , Pharmacology , Magnetics , Nanoparticles , U937 Cells , Xanthones , Pharmacology
12.
Journal of Experimental Hematology ; (6): 74-78, 2010.
Article in Chinese | WPRIM | ID: wpr-328569

ABSTRACT

This study was purposed to investigate the effect of a hypoxia-inducible factor inhibitor (YC-1) on expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) as well as induction of apoptosis in leukemic cell lines. RT-PCR was used to determine the levels of HIF-1alpha mRNA and VEGF mRNA in K562, U937 and Jurkat cells. After treatment of U937 cell with 4 micromol/L YC-1, cell apoptosis was assayed by DAPI staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining; the expression levels of HIF-1alpha mRNA and VEGF mRNA were measured with RT-PCR; the expression levels of HIF-1alpha, VEGF, BAX, BCL-2 and caspase-3 proteins were measured by Western blot. The results showed that HIF-1alpha mRNA and VEGF mRNA were expressed in all three leukemia cell lines. After treatment of U937 cell with 4 micromol/L YC-1 for 0, 8, 16 and 24 hours, the changes of morphologic features of U937 cells could be observed under fluorescent microscope and the apoptotic rates significantly increased in time-dependent manner, they were (4.87 +/- 0.70)%, (27.27 +/- 2.00)%, (51.53 +/- 2.81) and (60.5 +/- 3.20)% respectively, the expression levels of VEGF mRNA reduced, while the expression levels of HIF-1alpha mRNA had no obviously changes.Furthermore, the expression of HIF-1alpha, VEGF and BCL-2 decreased, while the expression of BAX and caspase-3 increased, the ratio of BAX/BCL-2 increased in time-dependent manner (r = 0.973, p < 0.01). It is concluded that HIF-1alpha mRNA and VEGF mRNA are all expressed in in K562, U937 and Jurkat cells, YC-1 has significant effect on down-regulating the protein expression of HIF-1alpha and VEGF, and induces the apoptosis in U937. The mechanism of apoptosis in leukemic cells may involve in up-regulating BAX/BCL-2 ratio and expression of protein caspase-3.


Subject(s)
Humans , Apoptosis , Cell Hypoxia , Gene Expression Regulation, Leukemic , Hypoxia-Inducible Factor 1, alpha Subunit , Metabolism , Indazoles , Pharmacology , Jurkat Cells , K562 Cells , U937 Cells , Vascular Endothelial Growth Factor A , Metabolism
13.
Journal of Experimental Hematology ; (6): 90-95, 2010.
Article in Chinese | WPRIM | ID: wpr-328566

ABSTRACT

This study was aimed to investigate the reversal effect of tyrosine kinase inhibitors (TKI) Imatinib and Nilotinib on multidrug-resistant cell line K562/A02. The expression levels of mdr-1 mRNA and bcr-abl mRNA were assayed by RT-PCR. The protein levels of P-glycoprotein (P-gp) and P210 were detected by Western blot. The daunorubicin (DNR) accumulation in K562/A02 cells were analyzed by flow cytometry (FCM). The results showed that the 0.0625 micromol/L Imatinib or 5 nmol/L Nilotinib alone had no cytotoxic effect on the inhibition of K562/A02 cells. When K562/A02 cells were treated with Imatinib or Nilotinib alone for 48 hours, the expressions of mdr-1 mRNA, der/abl mRNA, P-gp and P210 protein were all down-regulated, furthermore the effect of Nilotinib was stronger than that of Imatinib. The detection of fluorescence intensity revealed that the DNR concentration in K562/A02 cells treated with Imatinib or Nilotinib alone for 48 hours were 7.85% and 12.02% of K562 cells respectively. It is concluded that the tyrosine kinase inhibitors show great effect reversing drug resistance of cells, moreover, the effect of Nilotinib is stronger than that of Imatinib.


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Benzamides , Daunorubicin , Pharmacology , Doxorubicin , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Imatinib Mesylate , K562 Cells , Piperazines , Pharmacology , Protein Kinase Inhibitors , Pharmacology , Pyrimidines , Pharmacology
14.
Journal of Experimental Hematology ; (6): 127-131, 2010.
Article in Chinese | WPRIM | ID: wpr-328559

ABSTRACT

This study was purposed to construct and identify the short hairpin RNA (shRNA) eukaryotic expression vector for targeting gene mdr-1 which may play an important role in K562/A02. Short hairpin RNA (shRNA) aiming at the target sequence was to synthesized, the 3491-3509, 1539-1557and 3103-3121 nucleotide of mdr-1 mRNA were selected as targets. The selected nucleotides were cloned in the plasmid pGCSilencer-U6-neo-GFP respectively, and the resultant recombinant plasmids were named as pGY1-1, pGY1-2 and pGY1-3. The sequences of the recombinant plasmids were identified by DNA sequencing and PCR electrophoresis. The recombinant plasmids were transfected into the cell line K562/A02 by lipofection. After being transfected for 48 hours, the inhibition of mdr-1 mRNA was detected by real time-PCR, and P-gp expression was detected by Western blot. The results showed that the specific oligonucleotide was cloned into the vector successfully, and the expression of mdr-1 mRNA and P-gp in K562/A02 cells was reduced after transfecting the recombinant plasmid, as compared to the control group. It is concluded that the shRNA eukaryotic expression vector has been successfully established which can inhibit the expression of mdr-1 mRNA, setting up the basis to futher explore the effects of mdr-1 on cell line of K562/A02.


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Genetics , Drug Resistance, Multiple , Genetics , Drug Resistance, Neoplasm , Genetics , Gene Expression , Genetic Vectors , K562 Cells , Plasmids , RNA, Small Interfering , Genetics , Transfection
15.
Chinese Journal of Hematology ; (12): 385-388, 2010.
Article in Chinese | WPRIM | ID: wpr-353587

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the reversible effect of nilotinib, BrTet (5-bromotetrandrine) and their combination on multidrug resistance cell line K562/A02 and its mechanism.</p><p><b>METHODS</b>Cell proliferation inhibition was assessed by MTT method and cell apoptosis by flow cytometry (FCM). The expression of mdr1 mRNA was determined by RT-PCR, and the expression of P-gp was assessed by Western blot.</p><p><b>RESULTS</b>After 48 h 5 nmol/L nilotinib or 0.5 µmol/L BrTet treatment, IC(50) of daunorubicin (DNR) to K562/A02 was 4.52 mg/L or 5.41 mg/L respectively; While on combinative treatment, its IC(50) decreased to 2.98 mg/L. Nilotinib or BrTet alone was not able to increase the DNR induced apoptosis rate of K562/A02 cell (P > 0.05), while on combination treatment the apoptosis rate increased remarkably. After 48 h 5 nmol/L nilotinib or 0.5 µmol/L BrTet treatment alone, gray-scale value of mdr1 mRNA was 0.48 ± 0.04 or 0.64 ± 0.01, respectively; while on combinative treatment the value decreased to 0.35 ± 0.04. The P-gp expression level in K562/A02 cells was 0.61 ± 0.05, or 0.52 ± 0.02 when treated with 5 nmol/L nilotinib or 0.5 µmol/L BrTet alone for 48 h, but on combination treatment, the level decreased to 0.44 ± 0.03.</p><p><b>CONCLUSION</b>Nilotinib or BrTet alone can partially reverse drug resistance of K562/A02 cells. The mechanism may be associated with the decrease of mdr1 mRNA and P-gp expression and increase of the apoptosis rate. And there is a synergistic action with these two agants in combination.</p>


Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B, Member 1 , Metabolism , Daunorubicin , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , K562 Cells
16.
Journal of Experimental Hematology ; (6): 54-59, 2009.
Article in English | WPRIM | ID: wpr-302198

ABSTRACT

The aim of this study was to investigate the potential benefit of combination therapy with magnetic nanoparticle of Fe(3)O(4) and 5-Bromotetrandrine (5-BrTet) on chronic leukemia. The apoptosis was detected by flow cytometry (FCM), Wright staining and light microscope; the expressions of BAX and BCL-2 were measured by Western blot. The results showed that combination of daunorubicin (DNR) with either MNP (Fe(3)O(4)) or 5-BrTet exerted a potent cytotoxic effect on K562/A02 cells, while MNP (Fe(3)O(4)) and 5-BrTet co-treatment could synergistically enhance DNR-induced apoptosis. After treated with this regimen, the typical apoptotic morphological features were found in K562/A02 cells; the expression level of BCL-2 decreased and BAX increased markedly. It is concluded that MNP (Fe(3)O(4)) or 5-BrTet with DNR can induce apoptosis in K562/A02 cells, and they show distinct synergism when used together. The down-regulation of BCL-2 and the up-regulation of BAX may play important roles.


Subject(s)
Humans , Apoptosis , Benzylisoquinolines , Pharmacology , Daunorubicin , Pharmacology , Down-Regulation , Ferric Compounds , Gene Expression Regulation, Leukemic , K562 Cells , Nanoparticles , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Up-Regulation , bcl-2-Associated X Protein , Metabolism
17.
Journal of Experimental Hematology ; (6): 60-64, 2009.
Article in English | WPRIM | ID: wpr-302197

ABSTRACT

This study was aimed to investigate the reversal effect of 5-bromotetrandrine (5-BrTet) and magnetic nanoparticle of Fe(3)O(4) (Fe(3)O(4)-MNPs) combined with DNR in vivo. The xenograft leukemia model with stable multiple drug resistance in nude mice was established. The two sub-clones of K562 and K562/A02 cells were respectively inoculated subcutaneously into back of athymic nude mice (1 x 10(7) cells/each) to establish the leukemia xenograft models. Drug resistant and the sensitive tumor-bearing nude mice were both assigned randomly into 5 groups: group A was treated with NS; group B was treated with DNR; group C was treated with nanoparticle of Fe(3)O(4) combined with DNR; group D was treated with 5-BrTet combined with DNR; group E was treated with 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR. The incidence of tumor formation, growth characteristics, weight and volume of tumor were observed. The histopathologic examination of tumors and organs were carried out. The protein levels of BCL-2, BAX, and Caspase-3 in resistant tumors were detected by Western blot. The results indicated that 5-BrTet and magnetic nanoparticle of Fe(3)O(4) combined with DNR significantly suppressed growth of K562/A02 cell xenograft tumor, histopathologic examination of tumors showed the tumors necrosis obviously. Application of 5-BrTet and magnetic nanoparticle of Fe(3)O(4) inhibited the expression of BCL-2 protein and up-regulated the expression of BAX, and Caspase-3 protein in K562/A02 cell xenograft tumor. It is concluded that 5-bromotetrandrine and magnetic nanoparticle of Fe(3)O(4) combined with DNR have significant tumor-suppressing effect on MDR leukemia cell xenograft model.


Subject(s)
Animals , Female , Humans , Mice , Antineoplastic Agents , Pharmacology , Benzylisoquinolines , Pharmacology , Daunorubicin , Pharmacology , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Synergism , Ferric Compounds , K562 Cells , Mice, Inbred BALB C , Mice, Nude , Nanoparticles , Xenograft Model Antitumor Assays
18.
Journal of Experimental Hematology ; (6): 345-351, 2009.
Article in English | WPRIM | ID: wpr-302135

ABSTRACT

Multidrug resistance (MDR) plays a major role in the failure of cancer chemotherapy. Since Fe(3)O(4)-magnetic nanoparticle loaded with daunorubicin (DNR) can overcome multidrug-resistance of K562 cells in vitro, the effect of Fe(3)O(4)-magnetic nanoparticle loaded with DNR on multidrug-resistant K562 cells was studied in vivo, the K562-n and its MDR counterpart K562-n/VCR cells were inoculated subcutaneously into both sides of the back of nude mice to establish a human leukemia xenograft model. The mice were randomly divided into group A receiving normal saline, group B receiving DNR, group C receiving Fe(3)O(4)-magnetic nanoparticle, group D receiving Fe(3)O(4)-magnetic nanoparticle loaded with DNR and group E receiving Fe(3)O(4)-magnetic nanoparticle containing DNR with a magnetic field built on the surface of the tumor tissue. The tumor volume was measured on the day 1, 5, 9, 13, 17 and 21 after the first treatment. Tumor tissues were isolated for examination of the expression of mdr-1 by reverse transcription polymerase chain reaction and Western blotting. The results showed that for K562-n/VCR tumor, the tumor volume was markedly lower in groups D and E than that in groups A, B and C. Pathological observation revealed that the tumor cells of group A and B grew well, some disseminated necrosis and some cells with karyorrhexis and karyopyknosis existed in group C. However, significant fracture, necrosis of cell and subsequently fibrosis were seen in group D and E. The transcription of mdr-1 gene in groups D and E was significantly lower than that in groups A, B and C (group D and E vs group A, B or C, p < 0.05). However, there were no differences about the protein expression of P-gp between these groups. The tumor volume of K562-n in groups C, D and E was markedly lower than that in groups A and B (group C, D and E vs group A or B, p < 0.05). Pathological observation showed that the tumor cell of group A and B grew well, and no obvious necrosis was observed. Significant fracture, necrosis of cell and subsequently fibrosis were seen in group C, D and E. It is concluded that DNR-loaded Fe(3)O(4) magnetic nanoparticles can suppress the growth of the MDR K562-n/VCR tumor in vivo, but can not further enhance its efficacy on the sensitive K562-n tumor as compared to DNR alone. The additional external magnetic field failed to further improve the antitumor effect in vivo.


Subject(s)
Animals , Female , Humans , Mice , Daunorubicin , Pharmacology , Therapeutic Uses , Drug Carriers , Drug Resistance, Multiple , Drug Resistance, Neoplasm , K562 Cells , Leukemia , Drug Therapy , Magnetics , Mice, Inbred BALB C , Mice, Nude , Nanoparticles , Treatment Outcome , Xenograft Model Antitumor Assays
19.
Chinese Journal of Medical Genetics ; (6): 400-405, 2009.
Article in Chinese | WPRIM | ID: wpr-349067

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02.</p><p><b>METHODS</b>The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA (shRNA) or random-sequence (HK) were transfected into K562/A02 cells by lipofectamine mediation. cDNA microarray was performed to explore the alteration of gene expression profile when YB-1 gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). The proliferative ability of the cells was determined by methyl thiazolyltetrazolium (MTT) assay and cell cycle analysis. Cell apoptosis was assayed by Annexin V-FITC/PI double labeled flow cytometry.</p><p><b>RESULTS</b>The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47,000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT-PCR in K562/A02-YBX13 cells. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1, lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annexin V+ ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 micromol/L of As2O3.</p><p><b>CONCLUSION</b>Down-regulation of YB-1 gene by shRNA-YB-1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.</p>


Subject(s)
Humans , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , DNA-Binding Proteins , Genetics , Metabolism , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Leukemia , Genetics , Metabolism , Nuclear Proteins , Genetics , Metabolism , RNA, Small Interfering , Genetics , Y-Box-Binding Protein 1
20.
Journal of Experimental Hematology ; (6): 1413-1418, 2009.
Article in Chinese | WPRIM | ID: wpr-328630

ABSTRACT

This study was purposed to investigate the inhibitory effect of short hairpin RNA (shRNA) on expression of vascular endothelial growth factor (VEGF) receptor flt-1 gene in leukemia cells line K562, and to explore the influence of shRNA invasive ability on leukemia cells and its mechanism. The recombinant eukaryotic expression plasmid containing flt-1 shRNA gene was transfected into K562 cells by lipofectamine mediation and positive clones were screened by G418. shRNA gene in K562 cells was confirmed by PCR. RT-PCR and Western blot were employed to detect the expression of flt-1 mRNA and protein in leukemia cells. The invasive ability of K562 cells was studied by Boyden chamber invasion assay before and after flt-1 shRNA transfection. MMP-2 and MMP-9 mRNA expressions were detected by RT-PCR after transfection of the recombinant plasmid C1/U6/FltS2 into K562 cells through liposome. The results showed that the recombinant eukaryotic expression plasmid had been transfected into the human leukemia cell line K562 and positive clones had been screened by G418 for 2 weeks. PCR detection revealed the stable expression of the shRNA gene in K562 cells. Flt-1 gene and protein expressions were inhibited by plasmid-expressed shRNA after transfection of recombinant vetors C1/U6/FltS into K562 cells. The inhibitory efficiency of two different shRNA sequences targeting Flt-1 gene were 46.1% and 65.4% respectively. The expression of MMP-2 and MMP-9 mRNA decreased, and the mean invasion rate in C1/U6/fltS2-transfected K562 cells was lower than that in nontransfected cells. It is concluded that shRNA eukaryotic expression vector specific to VEGF receptor flt-1 gene can high efficiently be transfected into leukemia cell line K562, effectively inhibits the expression of flt-1 gene, weakens the in vitro invasive ability of leukemia cells and the expression levels of MMP-2 and MMP-9 mRNA, which suggests that the VEGF involves in the migration of leukemia cells by regulating the MMP-2 and MMP-9 through joints with the receptor.


Subject(s)
Humans , Gene Expression , Gene Expression Regulation, Neoplastic , Genetic Vectors , K562 Cells , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , RNA, Messenger , Genetics , RNA, Small Interfering , Transfection , Vascular Endothelial Growth Factor Receptor-1 , Genetics
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